RNA interference has emerged as a key pathway in gene regulation and the control of genome structure. It has also become a powerful tools for translating the lexicon that was revealed by genomic sequencing into a functional dictionary, complete with the definition of each gene. Understanding the biological relevance of the RNAi pathway and harnessing this pathway as a tool have both depended to a large degree on deciphering the biochemical basis of this gene silencing response. The work described in this application continues our studies of the mechanistic basis of RNAi through the application of structural biology, biochemistry and genetics in systems including Drosophila, mammals, C. elegans and plants. Specifically, the signals that determine how small RNAs enter RISC, the RNAi effector complex will be probed both from the perspective of the RNAs themselves and of the proteins that process them. The architecture and enzymology of RISC, the RNAi effector complex, will be examined during an RNA cleavage reaction, and the mechanism by which RISC can suppress protein synthesis in the absence of RNA cleavage will be sought. Finally, genetics and biochemistry will be applied to gain traction on the interactions between RNAi and chromatin structure that seem to be another evolutionary conserved effector arm of this flexible silencing pathway. These studies, if successful, will produce fundamental insights into a conserved silencing machinery with the additional benefit of honing tools that are being applied both for the discovery of new therapeutic targets and as potential therapeutics themselves. [unreadable] [unreadable] [unreadable]